Overweight and obesity among young, adult women are increasing problems in Sweden as in many other countries. The postpartum period may be a good opportunity to improve eating habits and lose weight in a sustainable manner. The aim was to make a cost-utility analysis of a dietary behavior modification treatment alongside usual care, compared to usual care alone, among lactating overweight and obese women.2, non-smoker, singleton term delivery, birth weight > 2500 g, intention to breastfeed for 6 mo and no diseases (mother and child). The women in the intervention group received 1.5 hour of individual counseling at study start and 1 hour at follow-up home visits after 6 weeks of intervention, with support through cell phone text messages every two wk. Dietary intervention aimed to reduce dietary intake by 500 kcal/day. The control group received usual care. Weight results have previously been reported. Here we report on analyses carried out during 2012-2013 of cost per quality adjusted life years (QALY), based on the changes in quality of life measured by EQ-5D-3 L and SF-6D. Likelihood of cost-effectiveness was calculated using Net Monetary Benefit method.This study was a cost-utility analysis based on a randomized controlled and longitudinal clinical diet intervention. Between 2007-2010, 68 women living in Sweden were, after baseline measurement at 8-12 weeks postpartum, randomly assigned to a 12-week dietary behavior modification treatment or control group. Inclusion criteria were: self-reported pre-pregnancy body mass index (BMI) 25-35 kg/mBased on conservative assumptions of no remaining effect after 1 year follow-up, the diet intervention was cost-effective. Costs per gained QALY were 8 643 – 9 758 USD. The likelihood for cost-effectiveness, considering a willingness to pay 50 000 USD for a QALY, was 87–93%.The diet intervention is cost-effective.NCT01343238 Registered April 27, 2011.ClinicalTrials.gov Identifier:The regional ethics committee in Gothenburg, Sweden, approved the study on November 15, 2006. Overweight and obesity among young, adult women are increasing problems in Sweden as in many other countries,2, and pWe conducted a randomized controlled study, consisting of a 12-week dietary modification program and a 9-month follow-up period, among women who were overweight or obese prior to pregnancy. The study showed significant and clinically relevant results, with a reduction of 2.7 BMI units in the dietary behavior modification treatment group compared to a reduction of 0.3 BMI units in the usual care control group . The weiThe aim of this study was to make a cost-utility analysis of a dietary behavior modification treatment, compared to usual care, among lactating overweight and obese women.2 before pregnancy, non-smoker, singleton term delivery, intention to breastfeed for 6 months, providing less than 20% of the child’s energy intake as a supplementary food, birth weight > 2 500 g, and no known disease in mother or child. Women with mild allergies and stable, medicated hypothyroidism were eligible. The regional ethics committee in Gothenburg, Sweden, approved the study. All participants provided written informed consent.Between 2007 and 2010, 76 women were recruited to the study from 15 maternity clinics in Gothenburg, Sweden. Inclusion criteria were: self-reported BMI 25-35 kg/mBaseline measurements were made 8-12 weeks after delivery. Out of the 76 recruited women, 68 women were, after baseline measurement, randomly assigned to either the dietary behavior modification treatment or the control group  with a nutrient composition according to the Nordic Nutrition Recommendations. The supWomen in the control group received usual care. They were asked not to engage in other lifestyle modification programs during the first 12 weeks of the study.Costs were during 2012-2013 calculated based on trial staff’s estimated time consumption per participant. In the calculation, national mean wages as well All costs were transformed from Swedish currency (SEK) to American dollars (USD) using the exchange rate 1 USD = 7.0 SEK. Costs were recalculated to year 2012 prices using the Swedish consumer price index. All effThe analysis in this study was a cost-utility analysis with a societal perspective, using gained quality adjusted life years (QALY) as the measure of health effects. Cost-efThe timeframe of the analysis is lifelong, but in the base case no effect of the intervention is assumed to remain after 1 year follow-up. The change from baseline to 1 year follow-up seems to have been progressive and is therefore unlikley to suddenly change. Hence, a mean of 1 year remaining effect may be more realistic than no effect after 1-year follow-up. In a sensitivity analysis, two alternative scenarios were calculated; all effects are lost 1 year and 3 years after the follow-up, respectively. Alternative cost effectiveness ratios were also calculated with reduced (50%) effect on QALY and doubled costs of the intervention.Cost-effectiveness is often calculated from the mean difference in costs and effects, and is calculated between two or more possible treatment options. The use of mean values for cost-effectiveness ratios is associated with uncertainty. In the current analysis, this uncertainty was handled with the Net Monetary Benefit method,20. The A scatter plot of 5 000 bootstrapped incremental cost-effectiveness ratios was created by repeatedly drawing a random sample with replacement using parameters estimated from the study. Individual values were used for gained QALY, and mean values were used for costs related to the intervention  that participants received. In such a way, the likelihood that the intervention was cost-effective using several thresholds of willingness to pay for a QALY was calculated. Further, mean Net Monetary Benefit and confidence intervals of Net Monetary Benefit were estimated for these threshold values.P-values <0.05 were regarded as statistically significant differences.Data were analyzed according to the intention-to-treat principle, with participants remaining in their original study groups. If data were missing, the last-observation-carried-forward method was used-23. PartOf 68 subjects included in the study, 57 completed the 1-year follow-up  (based on EQ-5D-3 L) and 0.044 (P = 0.03) (based on SF-6D) (Table P = 0.19) using the EQ-5D-3 L, and 0.031 (P = 0.07) using the SF-6D  and 9 758 USD (based on SF-6D) per gained QALY considering the follow-up year. With assumptions of 1 year remaining effect beyond the 1-year follow-up, the cost-effectiveness ratios were 3 477 and 4 088 USD per gained QALY, respectively. With the assumption of a 3-years remaining effect, the ratios were 1 644 and 1 939 USD per gained QALY, respectively.Net Monetary Benefits were higher for the intervention group than for the control group in all calculations, independent of time of remaining effect, value of a QALY and choice of QOL instrument. Net Moneary Benefts were significantly higher for the intervention group compared to those of the control group using calculations of QALYs based on SF-6D, a willingness to pay for a QALY of at least 100 000 USD, and with an assumption of remaining effect of at least 1 year. With the assumption of 3-year remaining effect, there were significant differences in Net Monetary Benefits between the groups also, with willingness to pay for a QALY set to 50 000 USD Table .The likelihood of cost-effectiveness was 0.52 – 0.95 considering follow-up year, when willingness to pay for a QALY was set to 10 000 – 100 000 USD  can be obtained also when gained QALY is a fifth of the base case. With halved gained QALY or with doubled cost, the cost-effectiveness ratios are still low (less than 20 000 USD/QALY).In this dietary weight loss intervention among overweight and obese lactating women, costs per gained QALY were 8 643 – 9 758 USD based on an assumption of no remaining effect after the follow-up year. With a willingness to pay of 50 000 USD for a QALY, the likelihood for cost-effectiveness was 87 – 93%.Long-term effects of weight loss programs have generally not been very successful, with half of the weight lost regained again during the first year of follow-up. In contThe approach in this analysis was to calculate the treatment effect as changes in QOL. Additionally, there may be preventive effects against diseases such as type 2 diabetes, heart diseases and osteoarthritis, with possibilities for even better cost-effectiveness ratios. Thus, our results likely underestimate the full cost-effectiveness of the intervention.Although not all beneficial effects are taken into account, the cost-effectiveness ratio in this study is well below usually accepted cost-effectiveness ratios. There is no formal level of acceptable cost of a QALY in the USA, although 50 000 USD and 100 000 USD are often used. There iTo our knowledge, this is the first analysis of cost-effectiveness of a certain diet intervention trial among overweight or obese women in the postpartum period. However, some analyses of interest have been published in similar fields. In 2011, a model analysis was made estimating cost-effectiveness of weight management interventions following pregnancy. Based oHealth economic analyses are often used as a basis for decisions on whether a method should become clinical standard or not. Hence, valid and reliable results are crucial. This requires analyses based on empirical data throughout the follow-up period, as was done in our case. Here, we present results on acceptable cost-effectiveness, which should be sufficient for making decisions on whether the method should be used as clinical standard or not. In addition, a preventive effect can be calculated from measured changes in lifestyle or medical risk markers among participants, in combination with epidemiological data on relationships between changes in these lifestyle factors and risk of diseases or premature death. Hence, the results presented here may underestimate the total expected health gains from the intervention.Of particular importance for determining the high quality of these analyses is our measurement of QOL. Two instruments were used, EQ-5D-3 L and SF-6D. EQ-5D-3 L is the most frequently used instrument in cost-effectiveness analyses, at least in Europe. However, this instrument may have ceiling-effect problems in such a young and healthy group of participants as ours. SF-6D, No measurements were made of participants’ costs for food and time spent cooking. Some studies have shown a healthy diet to be more costly while other studies deem this not to be the case,33. A stThe study was, in the original design, performed as a study with four study groups that also included physical activity interventions, both alone and in combination with diet intervention. The physical activity intervention was low targeted with a goal of 8 000 steps a day (which most of the participants reached) in the intervention group as well as in the control group. Hence, the physical activity intervention had no detectable impact on physical activity levels, energy expenditure, body weight or body composition.Two aspects will have a major impact on the cost-effectiveness: the amount of increase in QOL and how long the increase continues. We think our analyses have handled these aspects as thoroughly as possible. For even more secure results, larger studies with longer follow-up periods are needed. However, to date, our study has the longest follow-up available.According to this study, it may be cost-effective to promote weight loss by dietary changes among lactating overweight and obese women. Maternity leave usually entails changes in every day life and this period provides a window of opportunity to implement healthy and sustainable eating habits. Such programs could be offered within ordinary health care.BMI: Body mass index; EQ-5D-3 L: EuroQol 5 dimension questionnaire, 3 levels; GBP: Great Brittian pund; QALY: Quality adjusted life years; QOL: Quality of life; SEK: Swedish krona; SF-6D: Short form 6 dimension questionnaire; SF-36: Short form questionnaire; USD: United States dollar.The authors declare no conflicts of interest. The study was supported by grants from the Swedish Research Council (K2009-70X-21091-01-03) and the Swedish Council for Working Life and Social Research (2006-0339). The funders had no role in study design, data collection, analysis, decision to publish or preparation of the paper.AW, HB and FB: created the the study concept and design and were responsible for data collection; LH: Outlined the health economic analyzis, analyzed data and wrote the manuscript; AW, HB, FB and LH: interpreted results and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/38/prepub


FE) and photo-bias stability of the double-channel device were improved by increasing the thickness of the front IZO film (intt) compared to the single-ZTO-channel device. A high-mobility (approximately 32.3 cm2/Vs) ZTO/IZO transistor with excellent photo-bias stability was obtained from Sn doping of the front IZO layer. First-principles calculations revealed an increase in the formation energy of O vacancy defects in the Sn-doped IZO layer compared to the IZO layer. This observation suggests that the superior photo-bias stability of the double-channel device is due to the effect of Sn doping during thermal annealing. However, these improvements were observed only when intt was less than the critical thickness. The rationale for this observation is also discussed based on the oxygen vacancy defect model.This study examined the performance and photo-bias stability of double-channel ZnSnO/InZnO (ZTO/IZO) thin-film transistors. The field-effect mobility (μ Metal oxide thin-film transistors (TFTs) have attracted considerable attention for applications in the active-matrix (AM) backplanes of liquid crystal displays (LCDs) and organic light-emitting diode (OLED) displays due to their high mobility, low production cost, and low-temperature processing capability12362 or Al2O3, which were deposited by plasma-enhanced chemical vapor deposition (PECVD) or atomic layer deposition, were reported to effectively suppress the NBIS instability of the resulting metal oxide TFTs. This suppression was attributed to the large hole injection barrier (>2.0 eV) and low interfacial trap density of these insulatorsOV) defects is related to the intrinsic nature of metal oxide semiconductor materials. The incorporation of carrier suppressors, such as Zr, Hf, and Ga, to the relevant metal oxide semiconductor can help reduce the NBIS instability because these suppressors can effectively reduce the concentration of OV defects in the resulting channel layer. However, the field-effect mobility of the resulting oxide TFTs was adversely affected by the addition of such suppressors11High-quality gate insulators, such as SiOOV defects under In-rich conditionsThe incorporation of In cations to multi-component ZnO-based channels is one of best ways to enhance the field-effect mobility of the resulting oxide TFTs2 gate insulator on the mobility and positive-bias stress (PBS) instability of the resulting oxide TFTs. The improvement of both the mobility and PBS stability of IGZO/GZO TFTs was attributed to the localization effect of the conducting channel path, where the inserted conducting GZO layer reduced the screening length of the channel layer under an on-state current condition while suppressing the trap-induced PBS instability at the channel bulk and back surface regionSeveral studies have assessed the benefits of a double-channel structure15161718FE) of 32.3 cm2/Vs, a low subthreshold gate swing (SS) of 0.12 V/decade, a reasonable threshold voltage (Vth) of 0.5 V, a high Ion/off ratio of > 108, and the best NBIS stability. A depth profile analysis of the channel stack revealed the inter-diffusion of Sn and In atoms, which occurs during thermal annealing at 500°C. Therefore, the front IZO layer was doped with Sn cations. The microscopic origin for such improvement was examined by first-principles calculations based on density functional theory. The formation energy of oxygen vacancies for the IZO front layer substantially increased (by approximately 0.3 eV) due to Sn doping, which is responsible for the improvement in the NBIS stability of the double-channel device.In this study, double-channel oxide TFTs consisting of a back-layer ZTO film and front-layer IZO film were fabricated. The thickness-dependent performance and stability of the front IZO layer were examined experimentally. The optimum device was achieved for the 5-nm-thick IZO device, which exhibited a high field-effect mobility (μintt = 5.0 nm), and ZTO/IZO (intt = 6.3 nm) devices. μFE was determined by the maximum trans-conductance at a drain voltage (VDS) of 0.1 V, and Vth was determined from the gate voltage (VGS) required to produce a drain current of L/W × 10 nA at VDS = 5.1 V. The subthreshold gate swing (SS = dVGS/dlogIDS) was extracted from the linear portion of a plot of the log IDS versus VGS. For the first-series devices, the front IZO layer was prepared at an oxygen ratio ([O2]/[Ar + O2]) of 0.3. The single ZTO control device exhibited a μFE, SS, Vth, and Ion/off ratio of 28.8 cm2/Vs, 0.20 V/decade, 1.4 V, and 1.5 × 108, respectively. The transporting properties of the double-channel devices were improved substantially by increasing the thickness of the front IZO layer, as summarized in FE value obtained from the ZTO/IZO (intt = 6.3 nm) devices was 33.0 cm2/Vs. The enhanced transporting properties of the double-channel device were reflected in the superior output characteristics, as shown in th of the double-channel devices were displaced to the negative direction with increasing intt. This result indicates that the number of free electron carriers (Nd) in the channel layer is proportional to the thickness of the front IZO layer. The high mobility of the double-channel devices was attributed to the increasing In content caused by the intercalation of In atoms with a larger radius1th, the ZTO/IZO (intt = 5.0 nm) device was found to be optimal: a low voltage of 0.41 V is desirable for low-power-consumption AM panels. A similar behavior was observed at a higher oxygen ratio ([O2]/[Ar + O2]) of 0.4, as listed in it,max) according to the following equationB is Boltzmann's constant, T is the absolute temperature, q is the electron charge, and Ci is the gate capacitance per unit area. The Dit,max of the resulting devices decreased as intt was increased for an oxygen ratio of 0.3 and 0.4, respectively. This increase strongly suggests that the interfacial properties between the SiO2 gate insulator and oxide semiconductor channel layer were improved by inserting a front IZO layer. The physical implication will be discussed later in conjunction with the intt-dependent bias and photo-bias stability.The SS value of a given TFT device is related to the interfacial trap density (Dintt = 5.0 nm) devices. The devices were stressed under the following conditions: VGS and VDS were set to 20 V and 5.1 V, respectively, at room temperature, and the stress duration was 7,200 s. For the control device, a parallel Vth shift of 2.2 V to a higher voltage with increasing PBS time was observed without an accompanying change in μFE, SS, and the Ion/off ratio. The Vth stability of the IGZO TFTs under PBS was clearly improved by adopting a double-channel structure (see th shift was further suppressed to ~0.2 V for ZTO/IZO (intt = 6.3 and 8.3 nm) devices.The effects of the PBS and negative gate bias stress (NBS) on the transfer characteristics of both devices were investigated. ture see . It is nGS of −20 V and a stress duration of 7,200 s. Again, the NBS-induced variation was reduced significantly from −0.7 V (control device) to −0.3 V for the double-channel device, as shown in th variations of the ZTO/IZO devices prepared at an oxygen ratio of 0.3. The PBS-induced Vth displacement decreased significantly with increasing intt suggesting that the front IZO film provides a rapid carrier path and lower effective trap density. The Vth instability by the application of NBS was also suppressed by increasing intt, except for intt ≥ 6.3 nm.The effect of the channel structure on the NBS instability of the resulting TFTs was also investigated. The devices were stressed under a V2 was used under identical NBS conditions. Although the NBIS instability for each device was worse than the dark NBS instability, the changes in Vth by the application of NBIS were similar to those by NBS. When intt was less than the critical thickness (critt), the NBIS stability of the double-channel device improved with increasing intt. Here, critt is defined as the maximum thickness that results in higher mobility and better photo-bias stability compared to the single-channel device. For example, the negative Vth shift of the double-channel device (intt = 5 nm) was reduced from 12.5 V (single ZTO device) to 4.1 V (see intt ≤ critt) exhibited superior photo-bias stability, and the resulting oxide TFTs exhibited improved transporting properties compared to the control device. This result is difficult to understand because higher-In-containing oxide TFTs suffer from severe NBIS degradation due to the easy formation of oxygen vacancy defect centers. However, when intt > critt, the NBIS stability as well as NBS stability of the resulting double-channel device deteriorated abruptly. Therefore, the 6.3-nm-thick device fabricated at an oxygen ratio of 0.3 suffered from the most severe degradation (ΔVth = −15.2 V), which is larger than that of the ZTO control device, as shown in The photo-bias instability of the ZTO and ZTO/IZO devices was compared, as shown in .1 V see . The douintt = 5 nm) was much lower than that of the single-channel device. Here, the extracted DOS distributions are the summation of the bulk trap states (NSS) in the channel region and Nit at the channel/gate insulator interface. Because the thickness of the back ZTO channel was fixed to 35 nm for all devices, the NSS values for both devices would be comparable. Therefore, the In-rich composition can effectively eliminate the Nit at the IZO/gate insulator interface compared to those at the ZTO/gate insulator interface, which is consistent with the Nit,max variations extracted from the SS values.To obtain more insight into these anomalous behaviors, the DOS distributions of the devices in this study were calculated using the Meyer-Neldel ruleintt values. The MNR-derived DOS energy range was limited from the conduction band edge (EC) to (EC −0.4 eV), which corresponds to the tailing state of acceptor-like traps. Because the quasi-Fermi energy (EF) at the channel interface is positioned near the EC edge under the PBS condition, this DOS distribution directly affects the PBS instability, as discussed earlier. In contrast, NBS and NBIS instability mainly results from the VO deep state. Therefore, a complete understanding of photo-bias instability requires the comparative characterization of the VO density by X-ray photoelectron spectroscopy (XPS) and the theoretical electron calculation of VO defects.The PBS instability can be understood based on the carrier trapping mechanism. The amount of charge trapped into the channel bulk and at the channel/insulator interface will be proportional to the number of trap states regardless of the charge polarity. Therefore, the improvement in the PBS stability of the double structure devices can be attributed to the effective decrease in the interfacial trap density compared to that of the single-channel device. This inverse proportionality between the gate bias instability and DOS distribution was observed for all 2 stack. The Sn and In atoms in the channel stack region inter-diffused during thermal annealing for 1 h at 500°C, suggesting that the front IZO layer was doped with Sn cations. Pre-existing OV defects in the channel layer have a deleterious effect on the NBIS-induced instabilityOV) to the double-charged oxygen vacancy defects (OV+2), which results in the delocalized electrons in the conduction band. The influence of Sn on the oV formation energy was examined by first-principles calculations using the VIENNA ab initio simulation package (VASP)k-point mesh were used. For the exchange-correlation energy of electrons, the generalized-gradient approximation (GGA) + U calculation was used because it partially corrects the band-gap problem in GGA by decreasing the repulsion between oxygen p and metal d statesU parameters were set to 7.0, 3.5, and 8.0 eV for the In 4 d, Sn 4 d, and Zn 3 d states, respectively.Thus, the atomic distribution and chemical state of a ZTO/IZO film were analyzed by XPS. a-ITZO) compound with a stoichiometry of nIn:nSn:nZn = 1:1:1 was modeled by a first-principles molecular dynamics (MD) simulation. The eight formula units of In2Sn2Zn2O9 were distributed randomly into a cubic supercell. The structure was heated to 2,000 K for 5 ps, and then, the temperature was reduced rapidly to 300 K at a −300 K/ps quenching speed. The lattice parameters and atomic positions were fully relaxed, resulting in the final amorphous structure shown in The amorphous structure of the InSnZnO (f) for OV defects in the neutral state was calculated using the following formula: totE(OV) and OV, respectively, and μO is the chemical potential of oxygen under the given growth condition. In the present study, The formation energy (EfE(OV) depends strongly on the local environment of oxygenOV configurations exist in the amorphous structure. Therefore, 33 different OV configurations were calculated by removing an oxygen atom in the modeled a-ITZO and examining how the neighboring cation species affect the formation energy of the OV. In Sn). The formation energies increase substantially with increasing NSn, indicating that OV becomes difficult to form as more Sn atoms are introduced to the material. This dependence is consistent with the previous first-principles study that demonstrated the higher formation energy of OV in crystalline SnO2 compared to those of In2O3 and ZnOIn an amorphous multicomponent oxide, intt < critt) can be attributed to the suppression of OV defects in the front IZO layer by Sn doping and the reduced Nit density at the front layer/gate insulator interface.In addition, a recently proposed model on the peroxide formation through the hole creation inside the valence band may be valid in the present case and may also contribute to the observed photo instabilityintt > critt) suffers from inferior photo-bias stability compared to that of the single-channel device. OV is reduced due to the weak bonding of In and oxygen.The remaining question is why the thick front channel device  exhibited poorer NBIS stability due to the large OV concentration of the Sn-free front IZO layer. The extreme case corresponds to the single-IZO-channel device. The oxide TFTs with a 35-nm-thick single IZO channel did not exhibit any Ion/off modulation but instead exhibited simple metallic behavior. The Nd value of the 35-nm-thick IZO film was determined to be > 1020 cm−3 from the Hall effect measurement, which is consistent with the thick IZO film (intt > critt) with a high OV concentration.intt ≤ critt) and thick (intt > critt) IZO devices. There are two competing factors governing the PBS, NBS, and NBIS instability of the double-channel devices. One factor is the lower Dit,max between the Sn-doped IZO (IZTO) and SiO2 insulator compared to that between the ZTO and SiO2 insulator. The other factor is the creation of OV defects. For thin (intt ≤ critt) devices, the suppressed OV defect and lower Dit,max values are responsible for the improvement in the gate bias and photo-bias stability. However, thick (intt > critt) devices suffer from the abrupt creation of OV in the interfacial In-rich region due to the limited Sn diffusion to the IZO front layer, which leads to the deterioration of the NBS and NBIS stability.int dependency, the relative band structure for the ZTO/IZO/SiO2 stack was further analyzed by XP spectra using a monochromatic Al-Kα source. The XP valence band (VB) profiles near the bandgap for the ZTO, 5.0-nm-thick IZO, 6.3-nm-thick IZO, and SiO2 are depicted in 2, respectively. The band diagrams obtained from the VB spectra and optical bandgaps of the IZO and ZTO films are depicted in To confirm the proposed origin of the improved mobility and photo-bias stability with the strong tintt > critt) indeed has a high Nd due to the high OV concentration. The critt value of the double-channel TFTs clearly depends on the content of OV defect in a front layer. Indeed, the critt value for the double-channel TFTs increased from 5.0 to 6.5 nm when the oxygen ratio of O2/[O2 ± Ar] during the sputtering of the IZO layer increased from 0.3 to 0.4, as shown in O formation in the In-rich interfacial region due to the limited diffusion length of the Sn cation. In previous studies, the thickness of the front layer for the optimized double-channel device was in range of 3.5–5.0 nm18This remarkable difference suggests that the thicker IZO film  suffered from inferior NBS/NBIS stability and a large negative Vth displacement compared to those of the ZTO control device. This poor performance is attributed to the limited diffusion length of the Sn atoms compared to the thickness of the thick front IZO layer during thermal annealing. Therefore, careful control of the intt for the interfacial layer is essential for designing a double-channel device that exhibits improved performance and bias stability compared to the single-channel device. Overall, double-channel ZTO/IZO TFTs can be used as the backplane electronics for high-resolution and large-sized AMOLED and TFT-LCD displays.Double-channel ZTO/IZO TFTs exhibited improved mobility and photo-bias stability compared to single-channel ZTO TFTs when the front IZO thickness was less than the critical thickness. These intriguing properties were partially attributed to the reduced D2 thin film as a gate insulator was deposited onto a patterned Mo (200-nm-thick)/glass substrate by PECVD at 380°C. Front IZO thin films with intt values of 2.5–8.3 nm were deposited onto SiO2/Mo/glass substrates by DC sputtering. The working pressure was 0.26 Pa, and the relative oxygen ratio ([O2]/[Ar + O2]) was varied from 0.30 to 0.40. The DC power density of the InZnO (IZO) target (50 wt% In) was fixed to 2.2 W/cm2, whereas the deposition time was varied from 30 to 240 s. A 35-nm-thick ZTO film (back layer) was deposited onto an IZO/Mo/glass substrate. The oxygen flow rate of [O2]/[Ar + O2] was fixed to 0.10 for all devices. The Zn:Sn composition was approximately 50:50. The active channel was defined using a shadow mask during ZTO/IZO channel formation, and the ITO source/drain (S/D) electrode was deposited using the same sputtering system. The fabricated TFTs had a bottom gate structure with a channel width (W) and length (L) of 1,000 μm and 150 μm, respectively. The devices were annealed in air for 1 h at 500°C. The transfer characteristics of the oxide TFTs were measured using a Keithley 2636 Source Meter at room temperature. The film thickness was determined by ellipsometry and confirmed by X-ray reflectivity measurements. The cation compositions and chemical states of the ZTO and IZO films were examined by X-ray fluorescence spectroscopy  and XPS, respectively.A 120-nm-thick SiOH.Y.J., J.-U.B. and W.-S.S. designed this work. J.K.J. and S.H. wrote the manuscript. H.Y.J., A.Y.H. and D.-H.K. performed the experiments and electrical measurements. C.K.L. contributed to the XPS analysis. Y.K. and S.H. performed the theoretical calculations. All authors discussed the results and commented on the manuscript. The project was supervised by J.K.J.SI


Yersinia pestis synthesizes the attached biofilms in the flea proventriculus, which is important for the transmission of this pathogen by fleas. The hmsHFRS operons is responsible for the synthesis of exopolysaccharide (the major component of biofilm matrix), which is activated by the signaling molecule 3′, 5′-cyclic diguanylic acid (c-di-GMP) synthesized by the only two diguanylate cyclases HmsT, and YPO0449 (located in a putative operonYPO0450-0448).Y. pestis biofilm production in vitro and on nematode. Two distinct Fur box-like sequences were predicted within the promoter-proximal region of hmsT, suggesting that hmsT might be a direct Fur target. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that Fur specifically bound to the hmsT promoter-proximal region for repressing the hmsT transcription. In contrast, Fur had no regulatory effect on hmsHFRS and YPO0450-0448 at the transcriptional level. The detection of intracellular c-di-GMP levels revealed that Fur inhibited the c-di-GMP production.The phenotypic assays indicated that the transcriptional regulator Fur inhibited the Y. pestis Fur inhibits the c-di-GMP production through directly repressing the transcription of hmsT, and thus it acts as a repressor of biofilm formation. Since the relevant genetic contents for fur, hmsT, hmsHFRS, and YPO0450-0448 are extremely conserved between Y. pestis and typical Y. pseudotuberculosis, the above regulatory mechanisms can be applied to Y. pseudotuberculosis. Y. pestis is highly virulent to mammalians including humans, and causes systemic and fatal infections mainly manifested as bubonic, septicemic, and pneumonic plague. Y. pestis is primarily transmitted via the bite of an infected flea. Y. pestis synthesizes the attached biofilms in the flea proventriculus, making the blockage of fleas Yersinia biofilms are a population of bacterial colonies embedded in the self-synthesized extracellular matrix, and the matrix is primarily composed of exopolysaccharide that is the homopolymer of N-acetyl-D-glucosamine hmsHFRS operon is responsible for the synthesis and translocation of biofilm exopolysaccharide across the cell envelope, and all the four genes in this operon are required for the biofilm formation and for the flea blockage The Y. pestis to synthesize c-di-GMP, and both of them stimulate the Yersinia biofilm formation. The predominant effect of HmsT was on the in vitro biofilm formation, while the role of YPO0449 in the biofilm production is much greater in the flea than in vitroThe signaling molecule 3′, 5′-cyclic diguanylic acid (c-di-GMP) is a central positive activator of the enzymes catalyzing the production of biofilm exopolysaccharide Yersinia biofilm production in both nematode and flea models TAAGAAT-ATTCTTA is a 14 bp inverted repeat The Rcs phosphorelay system negatively controls Y. pestiscis-acting signals recognized by Fur AATGATAATNATTATCATT is a 9-1-9 inverted repeat.The ferric uptake regulator (Fur) is a predominant iron-regulating system in bacteria Y. pestis on the agar media, we found that the fur mutant exhibited a much more rugose and dry colony morphology relative to its parent strain, which promoted us to hypothesize the Fur-mediated repression of exopolysaccharide synthesis and biofilm production in Y. pestis (see below for details). In the present work, the detection of biofilms verified that Fur inhibited the Y. pestis biofilm production in vitro and on nematode. The subsequent gene regulation experiments disclosed that Fur specifically bound to the promoter-proximal region of hmsT for repressing the hmsT transcription, and yet, it had no regulatory effect on hmsHFRS and YPO0450-0448. In addition, the detection of intracellular levels of c-di-GMP revealed that Fur inhibited the c-di-GMP production. Therefore, Y. pestis Fur inhibited the c-di-GMP production through directly repressing the transcription of hmsT, and thus, it acted as a repressor of biofilm formation.During the general maintenance of Y. pestis biovar Microtus strain 201 is avirulent to humans but highly lethal to mice fur or the base pairs 146 to 468 of hmsS was replaced by the kanamycin resistance cassette by using the one-step inactivation method based on the lambda phage recombination system, to generate the fur or hmsS null mutant  of Y. pestis, as described previously hmsS lead to a biofilm-defective phenotype in Y. pestisΔhmsS was used as a reference biofilm-defective strain in this work.The wild-type (WT) fur coding region together with its promoter-proximal region (458 bp upstream the coding sequence) and transcriptional terminator (189 bp downstream) was cloned into the pACYC184 vector (GenBank accession number X06403) that harbors a chloramphenicol resistance gene. Upon being verified by DNA sequencing, the recombinant plasmid was introduced into Δfur, yielding the complemented mutant strain C-fur.A PCR-generated DNA fragment containing the Y. pestis cultivation, unless otherwise specifically indicated. For the general bacterial cultivation and maintenance, Y. pestis was cultivated in the Luria-Bertani (LB) broth or on the LB agar plate. For preparing the glycerol stocks of bacterial cells, a single colony was inoculated on the LB agar plate for further incubation for 1 to 2 d; the bacterial cells were washed into the LB broth at an optical density at 620 nm (OD620 ) of about 1.5, and stored with addition of 30% glycerol at −80°C. For primer extension or LacZ fusion, 200 µl of bacterial glycerol stocks were inoculated into 18 ml of fresh LB broth, and allowed to grow with shaking at 230 rpm to an OD620 of 0.4 to 0.5 prior to the bacterial harvest.The incubation temperature of 26°C was employed for the 32P] end-labeled reverse primer using a Primer Extension System (Promega) according to the manufacturer’s instructions. The same labeled primer was also used for sequencing with the fmol® DNA Cycle Sequencing System (Promega). The primer extension products and sequencing materials were concentrated and analyzed in a 6% polyacrylamide/8 M urea gel. The result was detected by autoradiography (Kodak film).Before bacterial harvest, double-volume RNAprotect Bacteria Reagent (Qiagen) was added immediately to each cell culture. Total bacterial RNAs were extracted using the TRIzol Reagent (Invitrogen) Y. pestis 201 genome DNA as template, and then cloned directionally into the HindIII-BamHI site of the transcriptional fusion vector pRW50 lacZ reporter gene. Correct clone was verified by DNA sequencing. Each Y. pestis strain tested was transformed with the recombinant plasmids. The empty plasmid was also introduced into each strain as negative control. The β-Galactosidase activity was measured on cellular extracts from cells cultivated as above by using the β-Galactosidase Enzyme Assay System (Promega) The promoter-proximal DNA region of each gene tested was prepared by PCR with the Takara ExTaq DNA polymerase by using fur was amplified from Y. pestis 201 and cloned directionally into the BamHI and HindIII site of plasmid pET28a (Novagen), which was verified by DNA sequencing. The recombinant plasmids encoding the His-Fur protein were transformed into Escherichia coli BL21 (DE3) cells (Novagen). Expression of His-Fur protein was induced by addition of 1 mM isopropyl-beta-D-thiogalactoside. The His-Fur protein was purified under native conditions with a QIAexpressionist™ Ni-NTA affinity chromatography (Qiagen). The purified, eluted protein was concentrated with the Amicon Ultra-15 (Millipore) to a final concentration of about 0.1 to 0.3 mg/ml in the storage buffer . The protein purity was verified by SDS-PAGE with silver staining. The purified protein was stored at −80°C.To prepare a His-Fur protein 32P] ATP and T4 polynucleotide kinase. DNA binding was performed in a 10 µl volume containing binding buffer , labeled DNA (1000 to 2000 c.p.m/µl) and increasing amounts of His-Fur. We still included two control reactions: one contained the specific DNA competitor , while the other was the non-specific protein competitor . After incubation at room temperature for 30 min, the products were loaded onto a native 4% (w/v) polyacrylamide gel and electrophoresed in 0.5×TB buffer containing 100 µM MnCl2 for 30 min at 220 V. Radioactive species were detected by autoradiography.For EMSA 32P-labeled forward or reverse one and its non-labeled counterpart. The PCR products were purified using Qiaquick columns (Qiagen). Increasing amount of purified His-protein was incubated with the labeled DNA fragment (2 to 5 pmol) for 30 min at room temperature in a final volume of 10 µl containing binding buffer same as EMSA. Before DNA digestion, 10 µl of Ca2+/Mg2+ solution (5 mM CaCl2 and 10 mM MgCl2) was added, followed by incubation for 1 min at room temperature. Then, the optimized RQ1 RNase-Free DNase I (Promega) was added to the reaction mixture, and the mixture was incubated at room temperature for 30 to 90 s. The cleavage reaction was stopped by adding 9 µl of the stop solution  followed by DNA extraction and precipitation. The partially digested DNA samples were then analyzed in a 6% polyacrylamide/8 M urea gel. Protected regions were identified by comparison with the sequence ladders. For sequencing, the fmol® DNA Cycle Sequencing System (Promega) was used. The result was detected by autoradiography (Kodak film).For DNase I footprinting 620 value of at least 1.0, stored at 4°C for cold shock for 8 to 12 h, and then diluted to an OD620 value of 0.8 with fresh LB broth. The diluted cultures were transferred into the 24-well tissue culture plates with 1 ml of cultures in each well, and allowed to grow at 230 rpm for 24 h. The media containing the planktonic cells were removed for determining the OD620 values. The well with the adherent biofilms was gently washed three times with 2 ml of H2O, and then incubated at 80°C for 15 min for the fixation of attached cells. The surface-attached cells were stained with 2 ml of 0.1% crystal violet for 15 min. The solution was removed, and the well was washed three times with 2 ml of H2O. Bound dye in the well was dissolved with 3 ml of dimethylsulfoxide. The OD570 values were recorded to indicate the crystal violet staining. The OD570/OD620 values were calculated to indicate the relative biofilm formation. The OD620 values were used for normalization to avoid the effect of growth rate and cell density.Two-hundred microlitre of bacterial glycerol stocks were spotted on the LB agar plate for further incubation for 1 to 2 d. The resulting bacterial cells were washed into the LB broth with an ODEscherichia coli OP50, a uracil auxotroph whose growth was limited on the NGM (Nematode Growth Medium) agar plates, were used as the standard foods for C. elegans. When the larvae or adults of C. elegans grow on the lawns of Y. pestis, this bacterium creates biofilms to cover primarily on the nematode head by blanketing the mouth and thus inhibiting the nematode feeding, which has been developed as a model for Yersinia biofilm research Y. pestis WT-GFP, Δfur-GFP, ΔhmsS-GFP, and E. coli OP50-GFP, respectively. To make the bacterial lawns, 200 µl of bacterial glycerol stocks were spotted on the LB agar plate for further incubation for 1 to 2 d. The resulting bacterial cells were washed into the LB broth with an OD620 value of at least 1.5, and aliquots of 300 µl were spotted on the LB agar plate for further incubation for 24 h. About 30 nematodes (adults or L4-stage larvae) were placed on each bacterial lawn expressing GFP, followed by incubation at 20°C for 12 h. The nematodes were suspended in the sterile M9 buffer  and then washed twice with M9 to remove planktonic bacteria. Worms was examined immediately by the epifluorescence microscopy.The lawns of biofilm-negative Aliquots of 5 µl of bacterial glycerol stocks were spotted on the LB plate, followed by the incubation for one week. The photograph of surface morphology of each bacterial colony was recorded.620 value of about 0.5, and then aliquots of 5 ml were harvested for the extraction of c-di-GMP with the extraction solvent . The chromatographic separation was performed on a Spark HPLC system equipped with a binary pump system and a 200 µl sample loop. The analyte detection was performed on an API 4000-QTRAP quadrupole mass spectrometer equipped with an electro spray ionization source (Applied Biosystems).The serially diluted water solutions of HPLC-grade c-di-GMP (KeraFAST) were used for determining the standard curves. The HPLC-grade xanthosine 3′,5′-cyclic monophosphate  was added as the internal standard into the c-di-GMP extract or standard solution at a final concentration of 50 ng/ml. Aliquots of 1 ml of bacterial cultures were harvest, and the amount of whole-cell protein was determined with a Micro BCA Protein Assay Kit (Thermo Scientific). The final c-di-GMP concentrations were expressed as pmol/mg of bacterial protein.The intracellular levels of c-di-GMP were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method as described previously t-test was performed to determine statistically significant differences, and P<0.01 was considered to indicate statistical significance. For primer extension, EMSA, and DNase I footprinting, the representative data from at least two independent biological replicates were shown.For phenotypic assays and LacZ fusion, experiments were performed with at least three independent bacterial cultures, and the values were expressed as mean ± standard deviation. Paired Student’s retrieve-seq’ program Y. pestis was used for the pattern matching within the target upstream DNA regions, by using the matrices-paster tool The 300 bp upstream regions of the genes tested  were retY. pestis cells tend to attach to the walls in vitro biofilms) can be detected with CV staining, which has been developed long time ago as a model for the determination of in vitro biofilms Δfur gave the normalized CV staining significantly greater than WT that was comparable to the complemented mutant C-fur, while the biofilm-negative strain ΔhmsS gave almost no CV straining  and E. coli OP50 (blank control) (control) .Y. pestis Fur acted as a repressor for the biofilm formation, most likely through inhibiting the production of biofilm exopolysaccharide.Taken together, hmsHFRS, hmsT, and YPO0450-0448. This analysis generated a weight score for each target promoter, and the higher score value indicated the higher probability of the Fur-promoter association hmsT rather than the remaining two  in WT or Δfur. A single transcriptional start site was detected to be located at the nucleotide A that was 128 bp upstream of hmsT, and thus, a single promoter was transcribed for hmsT. The primer extension assay also disclosed that the mRNA level of hmsT considerably enhanced in Δfur relative to WT.The primer extension experiments  were conhmsT, we constructed an hmsT::lacZ fusion vector, containing a 453 bp promoter-proximal region of hmsT and the promoterless lacZ, which was then transformed into WT or Δfur  and from 102 to 71 bp (site 1) upstream of hmsT, respectively. Both of them contained the Fur box-like sequences, and were considered as the Fur sites for hmsT  of the hmsHFRS and YPO0450-0448 operons. The primer extension  merged clone of the mild enteric pathogen Y. pseudotuberculosisY. pseudotuberculosis is transmitted by the food-borne route, while Y. pestis utilizes a radically different mechanism of transmission that rely primarily upon bite of fleas Y. pseudotuberculosis, but typical Y. pseudotuberculosis cannot synthesize adhesive biofilms on nematodes and make blockage in fleas Y. pseudotuberculosis NghA is a glycosyl hydrolase that cleaves the β-linked N-acetylglucosamine residues, and thus, it plays a key role in degrading the biofilm exopolysaccharide The hmsT , and thus, RcsAB acts as a repressor of Yersinia biofilm formation through inhibiting the production of both c-di-GMP and biofilm exopolysaccharide.The RcsAB box-like sequence can be predicted within the promoter-proximal regions of hmsT , hmsHFRS sequence , core promoter −10 and −35 elements for RNA polymerase recognition, and two cis-acting sites for Fur recognition, enabled us to depict the organization of Fur-dependent promoter of hmsT herein  or Δfur (b), and a standard sample of c-di-GMP (c) in water at a concentration of 0.3 nM were detected by HPLC-MS/MS -di-GMP c-di-GMP. cXMP was used as the internal standard at a concentration of 50 ng/ml.(JPG)Click here for additional data file.


A potential strategy for the diagnosis of lung cancer is to exploit the distinct metabolic signature of this disease by way of biomarkers found in different sample types. In this study, we investigated whether specific volatile organic compounds (VOCs) could be detected in the culture medium of the lung cancer cell line A549 in addition to the urine of mice implanted with A549 cells.Several VOCs were found at significantly increased or decreased concentrations in the headspace of the A549 cell culture medium as compared with the culture medium of two normal lung cell lines. We also analyzed the urine of mice implanted with A549 cells and several VOCs were also found to be significantly increased or decreased relative to urine obtained from control mice. It was also revealed that seven VOCs were found at increased concentrations in both sample types. These compounds were found to be dimethyl succinate, 2-pentanone, phenol, 2-methylpyrazine, 2-hexanone, 2-butanone and acetophenone.Both sample types produce distinct biomarker profiles, and VOCs have potential to distinguish between true- and false-positive screens for lung cancer. A 2008 report issued by the WHO , estimatThe ability to combine imaging techniques with other methodologies such as biomarkers is a strategy with the potential to enhance the detection of lung cancer -4. Altho13C-urea breath test for the detection of Helicobacter pylori [The analysis of exhaled breath for endogenous volatile organic compounds (VOCs) is one possible example of a non-invasive diagnostic assay that can be applied to cancer patients -9. Currer pylori ,11, and r pylori . AnotherSulfur-containing compounds such as ethylmercaptane, dimethylsulfide and dimethyldisulfide contribute to the characteristic odor of patients with liver cirrhosis . In contSeveral studies have shown that potential biomarkers for lung cancer are low molecular weight VOCs, which can be detected in the breath of lung cancer patients -24. For In addition to exhaled breath, urine is also considered to be a potential source of VOCs. However, one issue associated with the analysis of urine is the potential that VOCs detected in urine samples are derived from exogenous sources such as diet or the environment rather than as a result of the disease. For instance, Willis et al. reported that dogs could be trained to distinguish patients with bladder cancer on the basis of urine odor . Howeverin vitro [n-butyl acetate, acetonitrile, acrolein, methacrolein, 2-methylpropanal, 2-butanone, methyl tert-butyl ether and hexanal were all decreased [n-butyl acetate concentration which were found to be decreased [Nevertheless, the cellular and biochemical origin of endogenous VOCs that have potential as lung cancer biomarkers are not well understood. A number of articles have investigated the release of VOCs from human cancer cells in vitro ,30-32. Iecreased . In the ecreased , no VOCsIn the present study, we compared the VOCs released from the human lung adenocarcinoma cell line A549 to two normal lung cell lines, OUS-11 and WI-38 VA1 to determine whether any cancer specific compounds could be detected. We also investigated whether urine specific VOCs could be used to distinguish between tumor-bearing mice and healthy control mice, and whether there were any common compounds detected in both sample types.p-value < 0.10 and 1.5-fold higher or lower than average peak area of the normal control) were selected for identification. These ion peaks were summarized by deconvolution analysis of retention time and identified by spectral library match using the NIST'08 and Wiley library. However, identification of the VOCs was done only by means of spectral library matching (higher than 80% match) without confirmation of their retention times and comparison against a commercially available standard reagent. In this study, any peaks that could not be determined due to too low a library match were disregarded. The summary of determined VOCs by XCMS analysis are listed in Table m/z), comparing between the A549 and control cells. After a one week incubation period, there was a difference in the concentration of 31 compounds in the A549 cells compared with the control cells, whereby 18 compounds were found to be significant increased and 13 compounds were found to be significantly decreased (p < 0.10) (Table p < 0.10) (Table p < 0.10) . After the three week incubation period, many floating cells were observed and in all cell lines the culture medium was becoming amber. Thus, the culture conditions of the one and two week incubation periods ensured that the release of VOCs into the medium was mostly due to living cells. However, following three week incubation, cell viability diminished, indicating that released VOCs were not derived from the living cells. Typical total ion chromatograms (TICs) of VOCs measured in the medium samples of each cell line after one week incubation are shown in Figure To observe the proliferation of the A549 cells implanted into the mice, tumor size was measured Figure , with mep < 0.10 and characterized by a 1.5-fold higher or lower than average peak area of the control). Deconvolution analysis of the retention time of each peak allowed for the isolation of 76 VOCs that were significantly increased and six VOCs that were decreased. The isolated VOCs were picked up by similarity searching using the mass spectral library (NIST'08 and Wiley). Any VOC that could not be determined because a library match is too low was disregarded. Table m/z), comparing between the tumor-bearing and control mice. In the urine obtained from the tumor-bearing mice, the concentration of 43 compounds was different relative to the control group (p < 0.10), whereby 42 compounds were significantly increased, and one compound was significantly decreased.The pooled urine samples from the tumor-bearing n = 24) and control (n = 16) mice were analyzed by headspace solid-phase microextraction (HS-SPME) and gas chromatography time-of-flight mass spectrometry (GC-TOF MS). Two typical TICs of the VOCs obtained from the urine samples are shown in Figure 4 and conWe also investigated the compounds in more detail that were increased in both the one and two week culture medium samples (A549 cells), and urine of the tumor-bearing mice. Seven compounds were increased in all three samples. It was also discovered that dimethyl succinate, phenol, 2-methylpyrazine, 2-hexanone, 2-butanone, 2-pentanone and acetophenone could be identified not only by spectral library match using NIST'08 and Wiley library but also by matching with retention time and mass spectral of commercially available reagents (data not shown).m/z value. The calibration measurements were used to determine the limit of detection (LOD) and subsequent quantification (LOD) of seven VOCs. Results in Table 2 = 0.99). The concentration of each VOCs was then calculated using the respective standard curves. The quantitative findings, with accompanying statistical analysis, are summarized in Tables ANOVA.To determine the concentration of the seven VOCs, a standard curve was prepared with analysis by HS-SPME. The standard curves were induced using the peak area at most abundance p = 0.001), 2-pentanone (p = 0.015), 2-methylpyrazine (p = 0.012), dimethyl succinate (p = 0.023). Five VOCs in A549 cell medium were significantly increased in comparison to the VOCs in WI-38 VA13 cell medium; 2-pentanone (p = 0.001), 2-hexanone (p = 0.002), dimethyl succinate (p = 0.016), acetophenone (p = 0.001), phenol (p = 0.011). After two week incubation, five VOCs in A549 cell medium were significantly increased , 2-methylpyrazine was significantly decreased in comparison to VOCs in the DMEM. All seven VOCs in A549 cell medium were significantly increased in comparison to the VOCs in OUS-11 cell medium; 2-butanone (p < 0.001), 2-pentanone (p < 0.001), 2-hexanone (p = 0.008), 2-methylpyrazine (p < 0.001), dimethyl succinate (p < 0.001), acetphenone (p < 0.001) and phenol (p = 0.001). Five VOCs in A549 cell medium were significantly increased in comparison to the VOCs in WI-38 VA13 cell medium; 2-butanone (p = 0.001), 2-pentanone (p = 0.001), 2-hexanone (p = 0.039), dimethyl succinate (p < 0.001), acetphenone (p < 0.001).Summary of the concentration and significant difference of seven VOCs in culture medium were shown in Tables p = 0.02), 2-pentanone (p = 0.08), 2-hexanone (p = 0.02), 2-methylpyrazine (p = 0.03), dimethyl succinate (p = 0.03), acetphenone (p = 0.02) and phenol (p = 0.01).Summary of the concentration and significant difference of all seven VOCs in mice urine was shown in Table The ability to use the VOCs detected in the urine of patients with lung cancer as putative tumor markers may facilitate the rapid detection and non-invasive diagnosis of lung cancer. In this study, we identified VOCs derived from three cell lines  and from the urine of mice inoculated with the A549 human tumor cell line. The compounds were detected by HS-SPME and analyzed by GC-MS. The SPME method is simple and highly sensitive, and the in this study we used a SPME fiber (2 cm divinylbenzene/carboxen/polydimethylsiloxane: DVB/CAR/PDMS) which was the most absorbent as shown by the number of peaks which we were able to detect. Using this fiber, we analyzed the substances contained in the headspace of each sample.sec-butyl ester and 2,5-hexanedione. These include the plasticizer-like compounds . Thus, it is necessary to consider the possibility that exogenous compounds were also detected. Filipiak et al. also reported that the concentration of ethanol was significantly increased in A549 cells, although this finding was made under different experiment conditions [Several studies have also reported that VOCs are associated with lung cancer ,20-24. Fnditions . Howevernditions ,32. AddiWe created a mouse model of human lung cancer, and analyzed the VOCs obtained from the urine of these mice. When the urinary VOCs of the tumor-bearing mice were compared with of the control group differences in 43 compounds was observed (42 were increased and one was decreased). Matsumura et al. created a mouse lung cancer model, and identified the characteristics of some VOCs from the urine of these animals, including that 2-heptanone, 5-heptene-2-one were able to distinguish between the lung cancer group and the control group . Accordiin vivo model, it is difficult to nominate a culture period that closely mimics the characteristics of the cancer cells in the mice 26-30 days after transplantation. The conditions of the two week culture period are expected to be similar to the tumor-bearing mice, based on the idea that it takes a few days from seeding the cells until they are confluent. 2-Butanone, 2-pentanone, dimethyl succinate, and acetophenone in A549 culture medium were significantly increased in comparison to other samples including DMEM only. So these five VOCs considered suitable candidates for cancer biomarkers. Since 2-methylpyrazine and 2-hexanone are not likely to be released from A549 cancer cell, we currently made a conclusion to discard these compounds from candidates for biomarker. O'Nell et al. reported that 2-butanone and acetophenone can be detected in the exhaled breath of lung cancer patients [We also found that seven VOCs  were increased in both the urine of the tumor-bearing mice and the culture medium of the A549 cells. These compounds were identified not only by spectral library matching with NIST'08 and the Wiley library but also by matching the retention times and mass spectrum of commercially available reagents. However, with respect to which culture period is the optimal condition with which to compare with the patients . It has patients .Based on the results obtained from this study, it could be also suggested that some of the VOCs unique to lung cancer may also be detected in urine. Our results provide preliminary evidence that urinary biomarkers may also represent a feasible method for the early detection and non-invasive diagnosis of lung cancer.2. For the VOC measurements, the A549, OUS-11 and WI-38 VA13 cells were incubated for one, two and three-weeks after all cell lines grew as monolayer adherent on the surface of culture dishes. After this incubation period, the culture medium was collected and stored at -80°C until analysis.Three different human cell lines were used in the experiments: A549 (JCRB No. JCRB0076), OUS-11 (JCRB No. JCRB1034), WI-38 VA13 (JCRB No. JCRB9057). The A549 cells were originally isolated from a lung carcinoma of a 58-y-old man and are characterized by the presence of a mutated K-ras but a wild-type B-raf gene. The OUS-11 and WI-38 VA13 cells were used as normal control cells for the purposes of comparison and were derived from normal tissue of lung cancer patient and from a SV40 virus transformed lung fibroblast cell line, respectively. The cell lines were obtained from the Health Science Research Resource Bank . All cells have grown in DMEM high-glucose culture medium  supplemented with 10% fetal bovine serum , penicillin  and streptomycin . The cells were cultured in two culture dishes (100 mm × 20 mm) in 10 mL culture medium and every three days fresh medium was applied. For all experiments, the cells were cultured under standard conditions at 37°C in humidified atmosphere containing 5% CO8 cells/mL in PBS, and implanted subcutaneously into the right ventral flank area by injecting 0.1 mL of the suspension. The control mice were injected only with 0.1 mL PBS on the same position as tumor-bearing mice. The day of inoculation was defined as day 0. The mice were kept in glass-shielded metabolic cages  under sterile conditions and were supplied with radiation sterilized feed CE-2  and autoclaved drinking water, which was provided ad libitum. After transplantation, urine was collected from the mice daily, and stored at -80°C until time of analysis. Body weight and tumor size (the shortest and longest diameters) of each mouse was recorded every few days. Tumor size was calculated by multiplying the length of the shortest and longest diameters. The mice were sacrificed 30 days after transplantation. The urine samples were collected from 26 to 30 days after transplantation.Five-week-old female C.B-17/lcr-scidJcl mice  were separated into two groups, those bearing tumors (n = 24) and those used as controls (n = 16). To establish the human tumor-bearing model, A549 cancer cells were prepared at a concentration of 1.0 × 10All animals were treated in accordance with the institutional guidelines for the care and use of laboratory animals.g for 5 min and, the supernatant was used for the analysis. The VOCs from urine and cell culture medium were extracted by HS-SPME methods using of Combi-pal auto sampler . A 200 mL sample of the urine or culture medium was applied to a 2 mL crimp top vial and sealed by crimp cap. The vial was set on Combi-pal and equilibrated for 10 min at 45°C. The volatile compounds in the headspace were extracted by SPME fiber for 50 min at 45°C.Prior to the analysis of the urine and culture medium, we sought to determine the optimal suitable SPME fiber. Four types of SPME fibers  were compared with the chromatogram obtained from the analysis of urine samples. The results of this analysis, revealed the 2 cm 50/30 μm DVB/CAR/PDMS  fiber to be the optimal and this fiber was used for subsequent analyses. The urine and cell culture medium were centrifuged at 10,000 m/z 40 to m/z 500. Peak identification was accomplished through manual interpretation of spectra and matching against the mass spectral library ; mass spectral library of drugs, poisons, pesticides, pollutants and their metabolites Wiley, USA) and comparison with commercially available standard samples, which were purchased from the Tokyo Chemical Industry . Each medium sample was analyzed in triplicate.The GC-TOF MS system was composed of a 7890a GC  equipped with an auto sampler Combi-pal and GCT Premier  for time of flight mass spectrometry. The SPME fiber with absorbed volatile compounds was inserted into the injection port of the 7890a GC using the auto sampler and desorbed for 10 min at 240°C. The injection was pulsed splitless (closed 3 min) with a 0.75 mm liner. The GC-TOF MS system was equipped with an InertCap Pure-WAX T.L. column , which was used for separation and analysis of the desorbed volatiles. We employed the following chromatographic protocol for separation before MS analyses: 40°C for 5 min, then programmed at 3°C/min to 240°C with a 5 min hold at this final temperature. Column flow was constant at rate of 1 ml/min. The injection port was held at 240°C. Operating parameters for the mass spectrometer were as follows: the ion source temperature was 200°C, ionizing energy at 70 eV, scanning frequency was 0.2 s/spectrum from http://masspec.scripps.edu) [http://cran.r-project.org/). The significance levels of differences between groups were calculated using a student's t-test that come with XCMS software. We also determined that the "increased extracted ion peaks" had a p-value of < 0.10, and were 1.5 times higher than the averaged peak area of the other group. Of those peaks, a manual inspection of the EIC for each peak was made to validate the detected peak. Refine the VOC was performed by manual deconvolution based on retention time and peak shape of each increased extracted ion peak.The chromatographic peak areas were integrated using the MassLynx 4.1 . Genesis peak detection was applied for a list of putatively annotated ions. Detection and integration of generated ion peak from electron ionization were also performed using a XCMS software package version 1.16.3 (pps.edu) , runningp-values were obtained using ANOVA.The identified VOCs were quantified using commercially available reagents. For each of the reagents, stock solutions were made to a concentration of 100 mM  by dissolving them into 1 mL mixture of water and methanol (1/1 v/v). Calibration solutions of 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, and 10.0 μM were also made up. The standard curves were created based on the peak areas, which were obtained from HS-SPME GC-TOF MS analysis of the calibration solution. The data were analyzed in triplicate. The VOCs: volatile organic compounds; COPD: chronic obstructive lung disease; TIC: total ion chromatogram; HS-SPME: headspace solid-phase microextraction; GC-TOF MS: gas chromatography time-of-flight mass spectrometry; DVB/CAR/PDMS: divinylbenzene/carboxen/polydimethylsiloxane; CAR/PDMS: carboxen/polydimethylsiloxane; PDMS/DVB: polydimethylsiloxane/divinylbenzeneThe authors declare that they have no competing interests.YH carried out the experiments and participated in the design of the study. KS advised in the study design and helped to draft the manuscript. YB helped to draft the manuscript. HO, KY, and GKB conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


FTO rs9939609 increased plasma high sensitivity C-reactive protein (hs-CRP) levels independent of fatness. We aimed to investigate if this gene variant had fatness-independent effects on plasma hs-CRP and 10 additional circulating obesity-related adipokines throughout a broad range of body mass index (BMI) among Danish men.A recent study reported that the fatness associated A-allele of 2, all of whom were above the 99th percentile of this population). At an average age of 49 years (range: 39 through 65 years), 551 men, hereof 231 of the obese, were re-examined, including genotyping and measurement of the fasting circulating inflammatory markers hs-CRP, IL-1β, IL-6, IL-10, IL-18, mip1α, mip1β, sTNFα-R1, TGF-β, TNF-α and leptin. Men with known disease were excluded from the examination. All the inflammatory markers were log-transformed to approximate a normal distribution. Genotype-phenotype relationships were studied using linear regression analyses with the inflammatory markers as the response variable. Significant positive associations between hs-CRP, leptin and a broad range of BMI were observed, but the associations did not significantly differ across FTO rs9939609 genotype. There were no significant associations between the other inflammatory markers, FTO rs9939609 genotype or BMI, respectively.In a population of 362,200 young men, examined for military service between 1943 and 1977, two groups were identified: 1) a random 1% sample and 2) all obese men (BMI = 31.0 kg/mFTO rs9939609 A-allele on a series of inflammatory markers were observed in this cohort of healthy middle-aged men representing a broad range of fatness.No fatness-independent effects of the  Along with the rising prevalence of obesity worldwide FTO rs9939609 is the genetic variant most strongly associated with common obesity FTO rs9939609 A-allele causes variation in CRP levels independent of its effect on fatness There is an obvious lack in the understanding of causes behind and modifiers of this obesity-related inflammatory activity and environmental as well as genetic factors may be considered to play a role in the aetiology of this condition. Hitherto, the A-allele of the FTO rs9939609 is associated with a range of other inflammatory markers. The aim of the present study was to investigate whether the FTO rs9939609 A-allele has fatness-independent effects on circulating levels of the inflammatory markers hs-CRP, interleukin (IL)-1β, IL-6, IL-10, IL-18, tumor necrosis factor-alfa (TNF-α), soluble tumour necrosis factor α receptor antagonist (sTNFα-R1), transforming growth factor-beta (TGF-β), macrophage inflammatory proteins (MiP)-1α, and MiP-1β. Several of these markers are secreted from the adipose tissue, so-called adipokines, which also includes the non-inflammatory marker leptin. Leptin was included in the analyses as an adipocyte marker.In addition to CRP, it is thus of interest to investigate whether the 2  was sampled manually from the draft board files during the 1970s. The latter group represented all at least 35% overweight men according to a national standard in use at the time of sampling . This sampling design provided coverage of the entire distribution of BMI in the population with a 100-fold relative over-sampling of the obese, in order to allow particular focus on obesity and related aspects. All obese and half of the controls were invited to participate in the examination programme of the Copenhagen City Heart Study in 1981–83 and again in 1991–93. Details of these surveys are published elsewhere The participants included in this study were originally identified among a total of 362,200 Danish men who underwent the mandatory draft board examination in Copenhagen during 1943–1977 and in the remainder of Sjælland during 1964–1977 A last thorough whole-day examination was conducted in 1998–2000 among subjects who participated in the Copenhagen City Heart Study in 1991–93. Exclusion criteria were age above 65 years (to avoid bias by aging), residence too far away from Copenhagen to allow one-day examination, refusal in the past to participate in follow-up examinations, regular medication and known disease. Details of this survey is published elsewhere FTO rs9939609  was successful in 97% of the samples with a genotype error rate of 0.27% based on 1,464 duplicate samples. All genotype groups obeyed the Hardy–Weinberg equilibrium and the A-allele frequency was 0.41 in controls and 0.51 in obese individuals. Molecular genetic analysis, including genotyping of the FTO SNP rs9939609, was conducted on 551 men, hereof 231 of the originally obese. At present, the SNPs that are strongest associated with BMI belong to a linkage disequilibrium (LD) block encompassing parts of the first two introns of FTOGenotyping of Objective measures of height and weight were obtained FTO rs9939609 gave the best model fit in both the linear and logistic regression models as compared to dominant and recessive models.The inflammatory markers were modelled as the response variable in both the linear and the logistic regression analyses. In the linear regressions only detectable measurements were included in the analyses. The distribution of the inflammatory markers were visually tested via Q-Q plots, and as they all departed from a normal distribution , they were log(e)-transformed FTO rs9939609 and with the anthropometric obesity measures in our cohort, both in crude analyses, and when mutually adjusted. Then we investigated if an association between BMI and the inflammatory markers differed across FTO rs9939609 genotype. The results from the linear regressions are presented as effect estimates with 95% CI, which represent a factor of change in the inflammatory marker relative to the measurement unit in the predicting covariate . The results for the logistic regressions are presented as odds ratios (OR) with 95% CI. Interactions between BMI and FTO rs9939609, both modeled as continuous variables , were investigated. The likelihood ratio test assessed whether the model with the product term provided a better fit than the model without the product term. All models were adjusted for age. All analyses were two-tailed and a significance level was accepted at p<0.05. Analyses were carried out with Stata .Due to the skewed inflammatory markers their characteristics are presented as geometric means (which is the antilog of the arithmetic mean of the logged data) with 95% confidence intervals (CIs). After checking for linearity, all analyses were run for the obese and randomly selected men combined, which was possible because BMI was used only as an independent covariate. We investigated if hs-CRP and the 10 other adipokines were associated with the The Danish Data Protection Agency and the Ethical Committees of Copenhagen and Frederiksberg municipalities approved the study, which was in accordance with the Helsinki Declaration II. All participants signed written consent before participating.FTO genotype. Men belonging to the obese cohort were obviously heavier, had larger waist circumference and WHR than the randomly selected men. The obese were also younger, which reflects the increasing prevalence of obesity in more recent time 2 at the draft board examination. Using the TT genotype as the reference group we found that the OR for the TA genotype was 1.29 (95% CI: 0.87–1.93) and for the AA genotype 2.22 (95% CI: 1.38–3.57).In FTO genotype. For the adipokines IL-1β, IL-10, IL-18, mip1α, mip1β, sTNFα-R1, TGF-β, and TNF- α there were no association with either FTO rs9939609 or BMI  were observed both among the randomly sampled and the obese men  and 0.31 (p<0.001), respectively). Similarly, positive correlation between leptin and fat mass (kg) was observed  in the randomly sampled and 0.86 (p<0.001) in the obese). There were no correlations between IL-6 and fat mass (kg)  in the obese)).FTO genotype and the anthropometric obesity measures, respectively, are presented in FTO genotype and hs-CRP were positively associated, e.g. the hs-CRP level increased by 17% per additional A-allele (p = 0.05). However, this association disappeared when BMI and the other obesity markers were taken into account. Similarly, the leptin level increased by 10% (p = 0.04) per additional A-allele, but BMI, WHR and waist circumference accounted for this association. Further, positive associations were seen between BMI, WHR and waist (for given BMI) and hs-CRP and leptin, respectively, and these remained significant when FTO genotype was taken into account. As an example, hs-CRP level increased by 20% per 10% increase in BMI (p<0.001), and by 19% (p<0.001) when FTO was adjusted for. There were no association between IL-6 and FTO genotype and the anthropometric obesity measures, respectively. Furthermore, no interactions between BMI and FTO genotype in regard to hs-CRP, IL-6 and leptin, respectively, were observed , leaving all associations fairly unchanged.Finally, virtually the same pattern of results was obtained by logistic regression analyses of the dichotomized values of the inflammatory markers .FTO rs9939609 genotype. For IL-6, there was a non-significant tendency to lower levels by the FTO A-allele, i.e. opposite to the expectation, independent of the fatness variables. There were no associations between IL-1β, IL-10, IL-18, mip1α, mip1β, sTNFα-R1, TGF-β, and TNF-α and FTO genotype or BMI, respectively, in this cohort. Generally, the confidence intervals were so narrow that it seems fair to suggest that all together the FTO rs9939609 A-allele does not have important fatness-independent effects on systemic inflammatory markers or adipokines.In the present study of middle-aged Danish men positive associations between hs-CRP, leptin and BMI were observed. The associations did not differ across FTO rs9939609 A-allele and hs-CRP was observed in a recent German study 2 among men and 25.7 kg/m2 among women. We did not find an association between FTO rs9939609 and systemic inflammatory markers among generally healthy middle-aged men representing a broad range of BMI. The restriction in the inclusion of men to those without known diseases or regular medication may explain why we did not find an association between BMI and a range of adipokines generally known to associate with increased BMI FTO rs9939609 is only speculative. Regarding the circulating hormone leptin, a study on diabetics of both sexes found that FTO rs9939609 affected circulating leptin levels, yet the effect was accounted for by BMI FTO rs9939609 A-allele also did not have fatness-independent effects on the circulating levels of leptin. Thus, there is no reason to believe that the FTO rs9939609 A-allele has any functional impact on leptin in addition to the effect on fatness. Finally, we were surprised that the FTO A-allele tended to lower IL-6 levels, since we would have expected obesity to lead to increased IL-6 levels A fatness-independent association between the FTO rs9939609 had a fatness-independent effect on mortality and morbidity when analysed in a dominant model. We found that carriers of the A-allele had nearly twice the mortality compared to non-carriers irrespective of BMI FTO rs9939609 had more than twice the risk of a myocardial infarction or cardiovascular death compared with non-carriers, when adjusted for age, gender, BMI, smoking and history of myocardial infarction FTO rs9939609 A-allele on systemic inflammation.We have previously shown that FTO rs9939609 genotype are very unlikely. A limitation of our study relates to the fact that the biology of the FTO rs9939609 is not fully known. Further, the investigated SNP is located in the first intron of the gene, which according to the current knowledge may not be biologically functioning, so even though only speculative, there is a possibility that this SNP is in linkage disequilibrium with a functional variant within the FTO or nearby genetic regions There are several strengths of this study, but also limitations that should to be taken into consideration. The study is based on a unique, well-defined large background study population of white men with the obese and random sample derived from the same genetically quite homogenous population, hence eliminating population stratification. The study design and size of the study population implied that the confidence limits on the non-significant associations were fairly narrow, meaning that presence of any true major associations related to the FTO rs9939609 genotype. There were no associations between IL-1β, IL-6, IL-10, IL-18, mip1α, mip1β, sTNFα-R1, TGF-β, and TNF-α or FTO genotype and BMI, respectively, in this cohort. All together our results do not support the tentative evidence that the FTO rs9939609 A-allele have fatness-independent effects on systemic inflammation.In summary, we found positive associations between hs-CRP, leptin and BMI; however, the associations did not differ across Table S1Geometric mean  of the inflammatory markers according to FTO rs9939609 genotype for the two cohorts.(DOC)Click here for additional data file.Table S2FTO rs9939609 adjusted for age and body mass index.Odds ratios* with 95% CIs of the inflammatory markers per additional A-allele of (DOC)Click here for additional data file.


Musclin is a novel skeletal muscle-derived secretory factor found in the signal sequence trap of mouse skeletal muscle cDNAs. Musclin possesses a region homologous to the natriuretic peptide family. Thus, musclin is found to bind with the natriuretic peptide clearance receptors. However, the role of musclin in vascular regulation remains unclear. In this study, we aim to investigate the direct effect of musclin on vascular tone and to analyze its role in hypertension using the spontaneously hypertensive rats (SHR). In aortic strips isolated from SHR, musclin induced contractions in a dose-dependent manner. We found that the musclin-induced vasoconstriction was more marked in SHR than in normal rats (WKY). Moreover, this contraction was reduced by blockade of natriuretic peptide receptor C using the ab14355 antibody. Therefore, mediation of the natriuretic peptide receptor in musclin-induced vasoconstriction can be considered. In addition, similar to the natriuretic peptide receptor, expression of the musclin gene in blood vessels was higher in SHR than in WKY. Injection of musclin markedly increased the blood pressure in rats that can be inhibited by anti-musclin antibodies. Musclin-induced vasoconstriction was more pronounced in SHR than in WKY as in its expression. Taken together, these results suggest that musclin is involved in blood pressure regulation. The higher expression of musclin in hypertension indicates that musclin could be used as a new target for the treatment of hypertension in the future. Musclin is a novel muscle-derived secretory peptide found in the signal sequence trap of mouse skeletal muscle cDNAs. Musclin mRNA was almost exclusively expressed in the skeletal muscle of rodents and obesity models . The funMusclin contains a region homologous to the members of the natriuretic peptide (NP) family, as well as a KKKR putative serine protease cleavage site, which is also characteristic of NP proteins . The mamThe mechanistic involvement of NPR-C in the regulation of vascular functions has been documented. When physiologically activated, NPR-C seems to exert vascular protective effects, suggesting a direct antihypertensive role. Indeed, CNP/NPR-C signal has also been suggested as a fundamental pathway underlying the endothelium-derived hyperpolarizing factor-dependent regulation of vascular tone in rat mesenteric resistance arteries and in the coronary vasculature . TherefoHowever, little is known about the effect of musclin on vascular tone, especially in pathological conditions. In the present study, we investigated the reaction of musclin with aortic strips isolated from either the normal Wistar Kyoto rats (WKY) or the spontaneously hypertensive rats (SHR). In addition, a specific antibody was used to demonstrate the potential relationship between musclin and NPR-C. Further, we analyzed the differences in the gene expression of musclin and NPR-C between WKY and SHR. Finally, we injected musclin into normal rats to observe the increase in blood pressure and applied the musclin antibodies in SHR to find the recovery of blood pressure. Thus, in the present study, we identified the role of musclin in blood pressure regulation.Mouse musclin (SFSGFGSPLDRLSAGSVEHRGKQRKAVDHSKKRFGIPMDRIGRNRL-SSSRG) was synthesized through a solid-phase methodology by using 9-fluorenylmethyloxycarbonyl on an automated peptide synthesizer . The crude peptide was purified by reverse-phase HPLC  on a Mightysil RP-18 GP column . The homogeneity of the purified peptide was confirmed by analytical HPLC, MALDI-TOF mass spectrometry (MS), and amino acid analysis.AM). Food and water were available ad libitum. All the animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal experiments were approved by the Regional Ethics Committee for Animal Research in Chi-Mei Medical Center  and Kagoshima University .Male SHR and age-matched WKY, weighing 320–340 g, were obtained from the National Animal Center . They were maintained in a temperature-controlled room (25°C ± 1°C) under a 12:12 light-dark cycle . After dissection, the aorta was cut into strip segments approximately 5 mm long. The strips were then mounted on 37° C organ baths filled with 10 mL of oxygenated Krebs buffer  containing 135 mmol/L NaCl, 5 mmol/L KCl, 2.5 mmol/L CaCl2, 1.3 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 20 mmol/L NaHCO3, and 10 mmol/L d-glucose (pH 7.4). Each preparation was connected to strain gauges . Isometric tension was recorded by using a chart-software . The strips were mounted and allowed to stabilize for 1.5 h. The preparation was then gradually stretched to achieve an optimal resting tension of 0.5 g. The contractions were calculated as the percentage of the increase in vascular tone induced by treatment with potassium chloride (50 mmol/L).The rats were euthanized by decapitation under anesthesia with 3% isoflurane. We rapidly removed the aorta and placed the sample in oxygenated Krebs buffer , with a wait time of 15–20 min between each musclin dosing. Then, the increase in tonic contraction (vasoconstriction) was evaluated. Once the sample stabilized, oxygenated Krebs buffer was replaced, and then potassium chloride (50 mmol/L)  was added as a positive control. Vasoconstriction was expressed as the percentage of potassium chloride (50 mmol/L)-induced contraction, while the concentration–response curve was generated in a cumulative fashion.To exclude a possible role of the endothelium in musclin-induced vasoconstriction, the tests were conducted in endothelium-denuded preparations. The endothelium was removed by gently rubbing it against the teeth of a pair of forceps. Successful removal of the endothelium was confirmed by histological identification and failure of 1 µmol/L acetylcholine to relax the rings that had been pre-contracted with potassium chloride as described previously .4-23 , a ring deleted version of ANP, was used as a specific NPR-C receptor agonist. The specificity of anti-NPR-C antibody antibody was characterized in A7r5 cells by using the calcium influx assay method in our preliminary experiments. Since cANP has the similar molecular structure with musclin, we also compared the effect of cANP on vasoconstriction in SHR and WKY. Aortic strips were exposed to cANP at concentration of 0.1, 1, 10, 100 nmol/L, and the changes in vasoconstriction of cANP-treated samples in SHR were compared with those in WKY.Aortic strips were exposed to anti-NPR-C antibody (ab14355) at dilutions of 1:10000, 1:5000, and 1:2500  for 15 min before addition of musclin (0.01 nmol/L). The changes in vasoconstriction of musclin-treated samples were compared with those in vehicle (distilled water)-treated controls. cANP or ANPRat cell line for vascular smooth muscle cells (A7r5 cells)  were cultured in RPMI-1640 medium  supplemented with 10% fetal calf serum (FCS) , penicillin (100 IU/mL), streptomycin (100 mg/mL) , and amphotericin B . The cells were trypsinized (trypsin used was purchased from Gibco) and subcultured once a week, and the medium was changed every 3–4 days. For the experiments, the cells were seeded on round (25 mm diameter) sterile glass coverslips and cultured for 48–72 h in RPMI-1640 medium supplemented as mentioned above.2, 1.4 mM MgCl2, 11.5 mM glucose, 1.8 mM Na2HPO4, and 10 mM Hepes-Tris; next, 5 µM fura-2 was added to this solution; and then, the cells were incubated for 1 h in humidified atmosphere containing 5% CO2 and 95% air at 37° C. The cells were washed and incubated for further 30 min in PSS. The A7r5 cells were then inserted into a thermostated (37° C) cuvette containing 2 mL of calcium-free PSS and various doses of musclin or inhibitor as indicated. The fluorescence was continuously recorded using a fluorescence spectrofluorimeter . The values of intracellular calcium ([Ca2+]i) were calculated from the ratio R = F340/F380 by the formula [Ca2+]i = KdB (R −Rmin)/(Rmax − R), where Kd is 225 nM, F is the fluorescence measured at 340 nm and 380 nm, and B is the ratio of fluorescence of the free dye to that of the Ca2+-bound dye measured at 380 nm. Rmax and Rmin were determined in separate experiments by using musclin to equilibrate [Ca2+]i with ambient [Ca2+] (Rmax) and adding 0.1 mM MnCl2 and 1 mM EGTA (Rmin). Background auto-fluorescence was measured in unloaded cells and was subtracted from all measurements.The changes in the intracellular calcium concentration were detected using the fluorescent probe fura-2 . A7r5 ceWKY and SHR were anesthetized with phenobarbital (50 mg/kg i.p.). We separately administered musclin (0.05 mg/kg) and anti-musclin antibody FL-133  at 1:5000, 1:1000, or 1:500 dilutions to the tails of WKY and SHR by intravenous injection. Systolic blood pressure of anesthetized WKY or SHR was measured using the tail cuff method by a sphygmomanometer without animal heating . Blood pressure was measured at 5-min intervals after intravenous injection of musclin. Each value was presented as the average of 3 independent readings.g for 10 min at 4° C, and the pellets were resuspended in ice-cold homogenized buffer. Then, the blots were separately developed using anti-NPR-C antibodies (ab14355)  and anti-musclin antibodies (FL-133). The intensity of the blots incubated with actin (Millipore) was used as internal control. Blots were incubated with the appropriate peroxidase-conjugated secondary antibodies. After removal of the secondary antibody, the blots were washed and developed by autoradiography using the ECL-Western Blotting System . The densities of the obtained immunoblot bands at 48 KDa (outer membrane receptor [OMR]) and 43 KDa (actin) were quantified using the Gel-Pro Analyzer software 4.0 . In addition, the expression of NPR-C in the A7r5 cell line was detected by western blot analysis in the same manner.The expression of musclin and NPR-C was examined in the membrane protein fraction obtained from aorta homogenates. Aorta homogenates were centrifuged at 1,000 × p-value of 0.05 was considered statistically significant in Student t-test.All the values are presented as mean ± standard error of mean (SEM) for a given number of animals or samples. Statistical analysis was performed in SPSS. The statistically significant difference was evaluated using one-way ANOVA with Dunnett’s test compared against no antibody for each strain in the musclin-induced vasoconstriction and the inhibitory effect of the anti-NPR-C antibody on the calcium influx in A7r5 cells. 2-way repeated measures ANOVA for each strain in others. Musclin and NPR-C expression in aortic tissue used Student t-test. The difference was compared at the significance level of 2α=0.05 in ANOVA. A Vasoconstriction was induced in a concentration-dependent manner by musclin (0.01–10 nmol/L) in the aortic strips isolated from WKY or SHR. The response to musclin in SHR aorta was stronger than that in WKY . Aortic To elucidate the relationship between musclin and NPR-C, the aortic strips were pre-treated with ab14355 anti-NPR-C antibody at dilutions of 1:10000, 1:5000, and 1:2500 before treatment with buffer containing 0.01 nmol/L musclin. This pre-treatment led to a concentration-dependent attenuation of the musclin-induced vasoconstriction . The vasSuccessful removal of the endothelium was confirmed in each strip. Since the removal of the endothelium did not cause any effect, musclin-induced vasoconstriction was calculated as the percentage of potassium chloride-induced contraction (data not shown). In SHR aortic rings without endothelium, musclin-induced vasoconstriction was 82.16% ± 1.96%, which was not significantly different from that in aortic rings with endothelium . Similar results were observed in aortic rings isolated from WKY, for which vasoconstriction in the presence and absence of endothelium was 61.85% ± 0.83% and 63.96% ± 1.28% , respectively.Western blot analysis demonstrated the expression of NPR-C in the A7r5 cell line. The expression of NPR-C increased with increasing membrane protein amounts, and this increase was in multiples of 21.68 µg/mL .2+]i in vasoconstriction caused by cANP and musclin. After incubation with musclin, the concentration of [Ca2+]i in A7r5 cells significantly increased in a concentration-dependent manner. cANP also induced a similar response in A7r5 cells  treatment via injection into the tail vein . The incThe anti-musclin antibody FL-133 was used to investigate the effect of musclin on blood pressure in SHR. The solutions of FL-133 at 1:5000, 1:1000, or 1:500 dilutions were administered to SHR via intravenous injection. FL-133 at 1:5000 dilution did not induce a change in the blood pressure, and it was therefore taken as the control. Differently from the control, FL-133 at 1:1000 dilution decreased the blood pressure in SHR at 10 min after the injection  . FL-133 In the present study, we found that musclin induced vasoconstriction in aortic strips isolated from WKY or SHR in a concentration-dependent manner. The response in SHR was more marked than in WKY. In addition, we used the ab14355 anti-NPR-C antibody to abolish the vasoconstriction caused by musclin in order to highlight the mediation of NPR-C. The first (88LDRL91) and second (117MDRI120) NP-homologous regions of musclin are responsible for the cooperative high-affinity binding to NPR-C [2+-loaded calmodulin and endothelial nitric oxide synthase (eNOS) through the Gi protein [The vasoconstriction induced by musclin appeared to be calcium-dependent. The intracellular signals linked with the NPR-C receptor have been demonstrated in cardiovascular tissues and cells . Recentl protein . In part protein . Musclin protein . A7r5 ce protein . In the In SHR, both musclin and NPR-C expressions were higher than in WKY. The precise role of NPR-C in the development of hypertension is still unclear. NPR-C mRNA is found in atrial tissues, mesentery, placenta, lungs, kidneys, venous tissue ,17, and The main role of NPR-C is known to clear natriuretic peptides which would decrease blood pressure . The posThe maximal vasoconstriction induced by the highest concentration of musclin (10 nmol/L) did not differ between SHR and WKY. In addition, the anti-NPR-C antibody could only induce a partial inhibition of vasoconstriction induced by musclin. It seems that only a part of musclin could bind with NPR-C to induce partial activation of NPR-C. Since musclin is not a specific ligand of NPR-C, it is hard to follow the traditional ligand-receptor relationship. Furthermore, the vascular-wall abnormalities in SHR arteries may lead to less efficient vessel contraction than that in the WKY samples . Taken tIn the present study, we found a higher expression of musclin in SHR that has not been mentioned before. Moreover, the blood pressure was raised by injection of musclin into the rats. The decrease of blood pressure in SHR following anti-musclin antibody treatment showed the role of endogenous musclin in mediating the blood pressure regulation in SHR. However, the highest dose of the anti-musclin antibody showed a modest inhibition but not a complete blockade in the blood pressure of SHR. Therefore, other factors that might influence blood pressure regulation in SHR should be concerned and it needs to be investigated in the future. It seems necessary to generate tissue-specific musclin knockouts for the proper assessment of the functional role of musclin. Nevertheless, musclin really mediates blood pressure regulation, and the role of musclin in the pathogenesis of hypertension, at least in part, is identified in the present study using SHR.In the in vitro studies, we observed musclin could induce vasoconstriction in aortic strips and increase the calcium concentrations in A7r5 cells. Because both were obtained without natriuretic peptides involved, it is suggested that musclin could trigger blood vessel contraction. In in vivo, blood pressure was raised by the injection of musclin into rats, but this action of musclin was only partially reduced by NPR-C antibody ab14335. Thus, musclin may have its own specific receptor and also could partially activate NPR-C. However, musclin competes effectively with NPs for binding to NPR-C sites and lead to increase in local concentrations of natriuretic peptides, which would be supposed to decrease blood pressure . The pos